A new phenotype for a herbicide resistant mutant of Synechocystis 6714 with a high sensitivity to photoinhibition

Plant Science I I5 (1996)

165-174

A new phenotype for a herbicide resistant mutant of Synechocystis 6714 with a high sensitivity to photoinhibition Sabine Constant”, Irhe Perewoskaa, Ladislav Nedbalb, Teresa Mirandac, Anne-Lise Etienne”, Diana Kirilovskya,* *Phrstcn+kmon

et dynumiyue

des membranes 46 rue d’Ulm,

hlnstitufe

of Microbiology.

“.Sec.tion oj’Bi&ner~etiyue,

v~~e’tuies, URA IXiO,

CNRS, E&e

Acudemy of Sciences. 37981 Trebon. DBCM,

Not-mule Superieure.

75230 Puris Cedex 05. Frunce INRA. CEN-Sacluy,

Czech Republic

91190, G(f sur Yvette, Frunce

Received 26 September 1995; accepted I7 October 1995

Abstract

In this article, we describe the genotype and phenotype of a double mutant of Synechocystis PCC 6714 carrying A251V and S264A mutations in the Qn pocket of the Dt protein. The mutation A25lV confers an increased sensitivity to high light on the Photosystcm II (PS II). The A251 V mutation also results in a modification of electron transfer between QeA and Qu, a destabilization of the S2Q-r3 state and a destabilization of the St state due to a better accessibility of the oxygen evolving complex to cytosolic reduclants. The double mutant is more sensitive to high light than the simple mutant. The electron transfcr hetween Q-,,

and Qt3 is modified

in both mutants.

In contrast

to the single mutant,

in the A251V.

S264A

mutant,

the

the SZQmA states are stabilized and there are no modifications on the donor side of the Photosystem II. We conclude that there is no direct relationship between the St destabilization and the increased sensitivity to high light. The correS?Q-”

and

lation between the sensitivity Keywords:

Cyanobacteria;

of the PS II to high light and QeA, Q-B stabilities

Dl protein:

Photoinhibition;

Photosystem

1. Introduction

II; Synechocystis

Ret’s [ 1,2]). It contains the binding niche of the molecules which serve as a secondary electron acceptor of PS II (Qa). D, also provides some of the ligands of the manganese cluster involved in water oxidation. The plastoquinone molecule, Qa, which binds to a niche formed by a large hydrophilic loop connecting helices D and E of D,, on the lumenal side of the membrane, is a mobile two electron acceptor. QA, which is usually singly reduced, is bound to D2 another protein of the core of PS II that forms a heteroplastoquinone

The D, protein, one of the proteins at the core of Photosystem II (PS II), is involved in water oxidation and plastoquinone reduction (for recent reviews see

Ahbr~,vicrti,)rl.r: phcnyl)-

Chl,

I. I dimethylurea;

chlorophyll;

DCMU,

3-(3,4

acceptor of PS II; QB. second quinone acceptor of PS II.

* Corresponding

dichloro-

PS II. photosystem II; QA. first quinone

author. Fax: +33

c-mail: [email protected]

I

44323935;

is also uncertain.

016%9452/96/$ IS.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved I’ll: SO304-3X35(96)04342-7

166

S. Constant et al. /Plant

dimer with Dl. After primary charge separation, a very rapid reduction of the primary quinone acceptor (QA) occurs and then the electron is transferred to Qa. This reaction is reversible and in the dark there is an equilibrium between the states QeAQn and QAQ-n. After a second charge separation Qn receives a second electron and leaves its site as QnH2. It is then replaced by an oxidized quinone [3,4]. On the other side of the membrane, four positive charges are generated by successive photoreactions and are stored as oxidizing equivalents to oxidize two molecules of water to one oxygen molecule [5,6]. Consequently, the water-splitting complex can be in five different redox states (the S states) [6]. The oxygen is released during the transition from S3 to Sa in which S4 is a transient state with a lifetime of some milliseconds. The Qn pocket is also the binding domain of several classes of herbicides [7]. By competing with Qa, these molecules prevent the electron transfer between QA and Qn and thus inhibit photosynthesis [S]. It was shown that herbicide resistance results from mutations of one or two amino acids in the Qn pocket region. Therefore, a way to study the consequences of modifications of the structure of D, on the function of PS II is to use herbicide resistant mutants. Our group has studied the influence of mutations in the Qn pocket on the electron transfer within the PS II in Synechocystis, a strain of cyanobacteria [91I]. The effect generally observed was a lowering of the efficiency of electron transfer between QA and Qn attributed to a change in the apparent equilibrium constant between QaAQB and QAQ-n [9]. Two of our mutants also displayed an obvious modification of the oxygen evolving system [ 10, I I] demonstrating that specific mutations located in the Qn pocket have long range effect on the properties of the other end of the Dt protein. Exposure of photosynthetic organisms to high light intensity induces a quenching of fluorescence, an inhibition of the PS II activity and a specific damage and proteolysis of Di (photoinhibition) (for a recent review see Ref. [ 121). If exposure of photosynthetic cells to high light intensity is not too long, PS II activity can be restored by changing the damaged D, protein for a new one. We have also studied the influence of the mutations of D, on the light sensitivity of PS II [ 11,13-151. We have demonstrated that

Science 115 (1996) 165-174

the mutation A251V induces an increased high light sensitivity. Under a light stress, the cells carrying this mutation lost the ability to recover PS II activity sooner than wild-type cells. As the majority of herbicide resistant mutants, the single mutant A2.51V has a modified apparent equilibrium constant on the acceptor side; the amplitude of the slow phase of the fluorescence yield transient after a flash is larger and the S, state is destabilized [ 1 I]. However, the larger difference with the wild-type resides in the shape of oxygen oscillations. The maximum is shifted to the fourth flash. Analysis of the oxygen emission pattern repeatedly gave a significant increase of the concentration of S, in the dark adapted cells of the mutant. We believe that a long ranging effect of the mutation causing a conformational change in D, produces a better accessibility of the donor side to reductants present in whole cells [l I]. Addition of a second mutation, F211 S, to the change A251V further increases the sensitivity to photoinhibition, while decreasing the effects of A251V on the donor side of PS II. In this work, we describe the genotype and the phenotype of another double mutant of Synechocystis PCC 6714, DM35, which also presents a high sensitivity to high light. 2. Materials and methods 2.1. Strains, growth conditions and mutant isolation Wild-type and mutant cells of Synechocystis PCC 6714 were grown in the mineral medium described by Herdman et al. [16] with twice the concentration of nitrate. The cells were grown in a rotatory shaker at 34°C in a COz-enriched atmosphere under illumination from fluorescent light of about 70pE mm2 SK]. 2.2. psbA cloning and sequencing The isolation of genomic DNA from the DM35 mutant of Synechocystis 6714, the phage IEMBL3 library construction, the screening of this library and hybridation analysis were carried out as described by Perewoska et al. [ 1 l] The psbA2 copy from DM35 was identified with a radioactive psbA specific probe by screening a IEMBL, library, containing 15 kb BamHI DNA fragments. The 15 kb DNA fragment

167

S. Constant et ul. / Plunt Sclence 1 I5 (1996) 165-I 74

containing the psbA2 copy was subsequently digested with the restriction endonuclease KpnI obtaining 0.2, 0.4 and 0.7 kb DNA fragments which hybridized with the radioactive psbA probe. The different fragments were subcloned in Bluescript plasmids. The sequence coding for the QB pocket of D, in Synechocystis 67 14 is localized in the 0.7 KpnI fragment [ 171. Since all the mutations shown to confer herbicide resistance were found between amino acid residues 211 and 275, we decided to sequence the 0.7 kb fragment. Dideoxy chain termination sequence reactions on double-stranded DNA templates were performed according to Toneguzzo et al. [ 181 using a Sequenase Kit. Oligo-nucleotide sequencing primers were synthesized on a Milligen 7500 DNA synthesizer. 2.3. Photoinhibition

and recovery experiment

Cells of Synechocystis 6714 wild-type and M3.5 and DM35 mutants were harvested by centrifugation and resuspended in fresh medium containing 50 mM Hepes (pH 6.8) at a final concentration of 3Opg Chl/ml. The cell suspension (35 ml) was incubated at 21°C in a glass tube (3 cm diameter) refrigerated by cooled water and illuminated by one or four Atralux spots of 150 W (each giving an intensity of about IOOOpE m-l s-l). The cells were gently stirred by a magnetic bar. At 45 and 60 min, 10 ml cell solutions were transferred from high light to low light. For recovery, the cells were incubated at 34°C in a rotatory shaker under an illumination of 70pE m-2 s-l. Photoinhibition and recovery were followed by fluorescence measurements. The F,,, level was determined in the presence of 2 x 1O-5 M DCMU for M35 and wild-type and 2 x lO+ M Ioxynil for DM35. 2.4. Fluorescence

ofherbicide

measurements resistance

and determination

Chlorophyll ffuorescence induction during the first tens of milliseconds was monitored with a homemade fluorimeter. The fluorescence was excited with a tungsten lamp through CS 5-59 and CS 4-96 Corning filters. The fluorescence was detected in the red region through a CS 2-64 Corning filter and Wratten 90 filter. The recordings were made with a multi-

channel

analyzer.

The

cell

suspension

contained

about 1 pg ChVml. The inhibition of PS II electron transport from Q-* to QB resulting from the presence of increasing concentrations of herbicides was calculated by measuring the fluorescence level. The I,, shown in Table 1 is the concentration of the herbicide needed to obtain a value of 50 for the following ratio: F, - Fd F max- Fo. F, is the initial fluorescence at the onset of illumination of the sample; F,,, is the fluorescence level attained when a maximal concentration of Q-* is present; it was determined in the presence of a saturating concentration of DCMU (2 X 1O-5 M) for the wild-type and M35 and 2 X lo-” M Ioxynil for DM35; F, is the intermediary fluorescence level when non-saturating concentrations of herbicides lead to the closure of part of the centers. The fluorimeter used for the fluorescence decay kinetics was a prototype from Photon Systems International spol s.r.0. (Trebon, Czech Republic). Both actinic and detecting flashes were generated by LED arrays with maximum emission at 650 nm. The fluorescence was detected by a PIN photodiode at wavelengths above 690 nm. The energy of the 15~s square actinic flash was sufficient to saturate PS II of cyanobacteria. The sample was at a concentration of 2 pg Chl/ml. No signal averaging was necessary. The first point was recorded 100 ps after the flash and the timing between the detecting flashes was adjusted to the time domain studied. The timing of the experiment and the storage and treatment of the fluorescence signal were done by computer with adapted software. 2.5. Oxygen measurements The amount of oxygen produced per flash during a sequence of saturating flashes was measured at 22°C with a rate electrode equivalent to that described by Joliot and Joliot [28]. The short (5~s) saturating flashes were produced by a Strobotac (General Radio Company). The spacing between flashes was 0.5 s. Cells were resuspended in a medium containing 20 mM Hepes (pH 6.5), 100 mM KC1 and 5 mM MgCl* at a concentration of about 15Opg Chl/ml and they were dark adapted for 5 min at 22°C prior to each flash sequence (unless otherwise mentioned). The miss and double hit parameters

168

S. Consfunt el ul. / Plunt Science 115 (1996) 165-174

and the initial S,, and S, apparent values were deduced using the Sigma method developed by Lavorel

resistance were found between amino acid residues 21 I and 275 we decided to sequence a 0.7 kb KpnIKpnI fragment that contains a portion of the psbA gene coding from amino acid 180 to the COOHterminal of the D, protein. The analysis of the 0.7 kb fragment sequence of DM35 showed a double nucleotide change with respect to the wild-type. The nucleotide change GCC to GTC results in the modification of alanine 251 to valine also present in M35 and the nucleotide change TCT to GCT results in the replacement of serine 264 to alanine.

1191. 2.6. Thermoluminescence

measurements

Thermoluminescence was measured in a homebuilt apparatus as described by Ducruet and Miranda [20]. In this set-up dark-adapted samples (5 min) can be rapidly cooled to -40°C after saturating flash(es) are fired at - 10°C. During a slow continuous warming (O.S”C/min) of the sample, luminescence is emitted when the temperature is sufficient to provide the activation energy needed for the recombination. For S2QmA recombination studies, the sample was incubated with 5 X 10e5 M DCMU in the dark prior to the flash. A flash was given at -10°C and the sample was frozen rapidly in liquid nitrogen. The thermoluminescence signal observed upon heating the sample was analyzed as described earlier [20].

3.2. Photoinhibition During exposure of Synechocystis cells to high light, a specific quenching of FmaX followed by inhibition of the oxygen evolving activity is observed [ 13-151. This quenching can be reversed when cells are transferred to low light. During recovery, the damaged D, protein is replaced and the PS II activity recovered. We tested the behavior of wild-type and mutant cells under medium and high inhibitory light illumination and during the recovery process. Photoinhibition and recovery were followed by fluorescence measurements. The cells were exposed to medium inhibitory light (IOOOpE m-* s-l) at 22°C in the presence of lincomycin, a protein synthesis inhibitor, in order to avoid D, synthesis. Under these conditions, the decrease of F, was faster in the DM35 (A251V, S264A) mutant than in the wild-type (Fig. I). When the cells were exposed to a higher light intensity (40OOpE mm2 s-l) the rates of fluorescence quenching were similar in the mutant and the wild-type (data not shown). However, the DM35 (A251V, S264A) cells reach faster a state from which they are no longer able to recover. Fig. 2 shows that the wild-type was still able to recover after 60 min of photoinhibition while the re-

3. Results 3. I. Mutant selection, herbicide und sequencing

qf the psbA2

resistance,

cloning

gene

The spontaneous double mutant DM35 described in this paper was selected from the simple mutant M35 (A25 1V) of Synechocystis 67 14 in the presence of IO-’ M DCMU. While the M35 mutant is resistant to atrazine, metribuzin and ioxynil, DM35 is resistant to DCMU, metribuzin and atrazine (Table 1). The Synechocystis genome contains three copies of psbA: two homologous copies, psbA2 and psbA3 and a divergent copy psbA1. Only the copy psbA2 carries the mutations [21]. This copy was cloned and sequenced. Since all the mutations shown to confer herbicide

Table I 150 concentmtion of different herbicides in WT, M35 (A25 IV) and DM3.5 (A251V.

S264A) cells

determined by

fluorescence measurements

Atrazine

DCMU

Ioxynil

Metribuzin

(M)

(M)

(M)

(M)

WT

3 x 10-6

1,s x 10-7

8.0 x lOA

3x

M3S (A251V)

4 x 10-5

5.0 x 10-7

3.6 x 1O-5

3 x 10-4

3.6 x 1O-6

9.0 x 10-6

DM35

(A251V.

F21 IS)

>10-4

>10-3

104

S. Constant

et al. /Plant

Science 115 (1996) 165-l

169

74

oscillatory pattern is damped due to (double hits) or misses in each state 2 shows that the miss parameter creased in the M35 (A251V) and

double turnovers transition. Table was slightly inDM35 (A251V,

loo-

Elo-

ok--T--0

”

”

10

20

Time

”

30

I ”

40

of photoinhibition

“,

”

50

”

I

60

(min)

Fig. 1. Fv decrease during exposure of medium inhibitory light (1000pE

me2 s-t)

of cells of WT

(A) and DM35

(A251V.

S264A) (0).

0

30

60

90

Time of recovery

coveries of DM35 (A251V, S264A) and M3.5 (A25 1V) cells were severely impaired. Comparison of the recovery rates of 45 min photoinhibited cells indicated that M35 (A25 IV), the simple mutant, presented an intermediate sensitivity to high light (Fig.

120

180

150

(min)

2). 3.3. Oxygen emission Since So and S, are the only redox states of the water splitting complex present in the dark, a period of four oscillations in oxygen emission per flash is produced on dark adapted oxygen evolving systems illuminated by a train of saturating flashes. In most cases, the first maximum is on the third flash. This is attributed to larger concentrations of centers in the S, state than in the So state in the dark adapted systems. The main difference between the wild-type and the M3.5 (A251V) mutant resides in the shape of the oxygen oscillation. In this mutant, the first maximum was shifted to the fourth flash (Fig. 3) due to a larger concentration of So than the wild-type after 5 min of dark adaptation [ 1 l] (Table 2). The double mutant, DM35 (A251V, S264A), presented a maximum of oxygen emission in the third flash and a lower So concentration than the simple mutant. The oxygen

0

30

60

90

Time of recovery

120

150

(min)

Fig. 2. Recovery of Fv under low light intensity (70 mE m-* s-‘) in WT (A), M35 (A25lV)

(0)

and DM35

(A251V,

S264A)

(0)

cells previously photoinhibited during 45 min (A) or 60 min (B).

170

S. Constant et (11./Plant

Science I15 (I 996) 165-I 74

3.4. The stability of the S, QeB state The S2 state is deactivated in the dark by recombination with the negative charge stored in the acceptor side. After charge stabilization, the negative charge is shared between QA and Qa. The recombination S2QmA is more rapid and needs less energy than the recombination &Q-a. A destabilization of Q-a decreases the energy needed for the Z&Q-a recombination. We determined the rate of the back reaction &Q-a with the oxygen rate electrode by varying the time between the first and the subsequent flashes. The overall half time was 40 s for the wild-type, 20 s for M35 (A251V) and 80 s for DM35 (A251V, S264A). For thermoluminescence measurements, a dark adapted sample was rapidly cooled to -40°C after a saturating flash given at -10°C. During a slow and continuous warming of the sample, luminescence was emitted in the temperature range allowing charge recombination. The thermoluminescence Q band, obtained in the presence of DCMU, is due to the S2QwArecombination and the B band to the &Q-a (or &Q-a) recombination [22-231. The temperature corresponding to the maximum peak of the Q band was 2°C in the wild-type and 10°C in the DM35 (A251V, S264A) mutant (Fig. 4A,B). As already published for other strains of cyanobacteria [24], two components were necessary to correctly fit the Q band. The main peak was at 2°C in the wild-type and 7.5”C in the DM35 (A251V, S264A) mutant, respectively. The small one was at 14°C in wild-type cells and 24°C in DM35 (A251V, S264A) cells. These results suggest that the SZQeA

a, .-E .-I

E

0,6

a,

0,4

5l 0)

0.2

xx

t0

0.8 E 0.0 0

;

4 6

8 10 1; 14

Number of flash

0

2

4

6

8

IO

12

14

Number of flash

Table 2 Parameters 0

2

4

6

8

10

12

of oxygen sequence

14

Number of flash Fig. 3. Oxygen yield (in relative units) per flash during train of saturating flashes (5,~s) spaced by 0.5 s. These ments were carried out in WT (0). M35 (A251V) (U) and (A251V. S264A) (Cl) cells previously dark-adapted during

S264A) mutants, as in cells of other mutants modified Qn pocket [9].

a short experiDM 35 5 min.

with a

WT M35 (A251V) DM3S (A251V. F211S)

so(%)

Sl (%)

a

37 55 45

63 45 ss

0.08 0.12 0.11

The values of the oxygen sequence parameters were computed by the matrix analysis from recorded oxygen sequences on samples dark-adapted for 5 min. So, St, Concentrations in the darkadapted state; a, miss parameter.

S. Constanr et al. /Plant I

--‘--i--T-IA-

I

7

WT

Temperatcre

(’ C)

171

Science 115 (1996) 165-174

main peak at 41.5”C and two smaller components with maxima at 22°C and at 50°C. The component at 22°C that was quite important can be related to one of the components of the Q band or to a destabilized &Q-a state. Both the oxygen measurement and the shift of the main component of the B band towards higher temperatures in the mutant were indicative of a stabilization of &Q-a. In consequence, the emission at 22°C should be related to the S2QmArecombination. In the M35 (A25 1V) mutant, as already published [I I], the Q band was similar to that of the wild-type and the B band was shifted to lower temperatures (24°C) indicating a destabilization of the &Q-n state.

I 3-

A251V.

S264A

i:t

Temperature Temperature

Fig. 4. Thermoluminescence -10°C (A25 (-_)

/

in presence of lo3

IV.

S264A)

( cl

( C)

Q band after a saturating flash at M DCMU

for WT

(A) and DM35

(9) cells. This figure shows the measured signal

and the theorical curves (- - - - -) obtained by a computer

simulation program 1201.

B-

2

state is more stable in DM35 (A251V, S264A) than in wild-type cells. The B bands after one flash in the wild-type and the mutant were very different (Fig. 5A,B). In the wild-type, the B band could be simulated by a main component with maximum at 39°C and two very small components with maxima at 10°C most probably related to S,Q-, and at 50°C. The precise origin of the emission at 50°C remains uncertain, but it was proposed that it is related to the Tyr+oQ-A recombination (for a review see Ref. [25]). In the mutant, the B band with maximum at 42°C presented a big shoulder at 25°C. This band could be simulated by a

A251V,

S264A

a, 2 u” s c E

:,j

ii

( Temperature

Fig. 5. Thermoluminescence -10°C

in WT

(A) and DM35

(.C)

B band after a saturating flash at (A251V,

figure shows the signal measured (---)

S264A)

(9)

cells. This

and the theoretical curves

(- - - - -) obtained by a computer simulation program [20].

5. Cmstunt et al. /Plant Screnc~e 115 (1996) 16.5-174

3.5. Fluorescence

0

1

3

4

Time

0

/

,

,

10

20

30

Time

6

7

9

,

,

I

50

60

70

10

(msec)

40

(msec)

C

0 0

5

10

15

20

25

30

Time (SW)

Fig. 6. Fluorescence decay after one saturating flash in darkadapted WT (0) and DM35 (A251V, S264A) (a) cells. The fluorescence was recorded between 100~s and 10ms in (A); between 200~s and 75 ms in (B) and between 75 ms and 25 s in (0.

decq

The kinetics of Q-A reoxidation were followed by Chl fluorescence decay after one saturating flash. When QA is in its reduced form, the PS II reaction center is closed and there is no photochemical quenching When QA is in its oxidized form. the center is open and there is an efficient photochemical quenching. The decay of fluorescence after one saturating flash, corresponds to the electron transfer from QeA to Qa (or Q-a) (in the range of hundreds of microseconds), to the establishment of a quasi-steadystate level of Q-,,, (in the range of milliseconds) which depends on the redox couples QA/QAA and Qn/Q-n and which is influenced by binding and release of Qa (and maybe Q-a) to and from its site and to the recombination reactions between the positive charges accumulated in the Mn cluster and the negative charges accumulated in the acceptor side (in the range of seconds). We observed that the amplitudes of the slower phases were larger and their kinetics were slower in the mutant (A25lV, S264A) than in the wild-type (Fig. 6). No significant differences were observed in the range of microseconds (Fig. 6). 4. Discussion By sequencing the region of the psbA gene corresponding to the Qa pocket, we demonstrated that the D, protein of DM35 carries two mutations: A25 IV and S264A. The combination of these two mutations causes modifications of the electron transfer on the acceptor side of PS 11 and an increased sensitivity to light stress. Some of the modifications already present in the simple mutant (A25 1V) were even larger in the double mutant (A251V, S264A) (e.g. slow down of the reoxidation of Q-A, sensitivity to high light) while others were suppressed (e.g. modifications on the donor side). In 1993, Etienne and Kirilovsky 1271 proposed a classification of the effects of mutations in the D-E loop of the Dt protein of cyanobacteria in three categories: (1) mutations which confer herbicide resistance but do not markedly affect the electron transfer. (2) Mutations which confer herbicide resistance and a shift of the apparent equilibrium constant on the acceptor side favoring the presence of the electron on

S. Crmtunt

et ui. /Plant

QA. The S2Q-B state, but not the SZQ-A state, is destabilized in these mutants. (3) Mutations which generate changes on the electron transfer reactions on the donor and acceptor side of PS II. In this type of mutants we observed a destabilization of S, due to a better accessibility of this oxidized state to reductants. A destabilization of the SZQ-B state is also observed as in the previous category. Regarding the herbicide resistance and the decay kinetics of the transient fluorescence yield, the DM35 (A251V, S264A) mutant could be placed in the second category of mutants. However, apart from these the phenotype of DM3.5 (A251V, similarities, S264A) is different from that of other herbicideresistant mutants. Measurements of thermoluminescence and oxygen evolution showed a stabilization of the S2QeA and !&Q-a states in DM35 (A251V. S264A) by contrast with the previously described mutants. Recently, in collaboration with Dr P. Mgenptia and co-workers [24], we studied the PS II activity of a site-specific mutant of Synechocystis 6803, CA 1, which presents a deletion of the three glutamates 242-244 and a substitution of glutamineby histidine in the Qr, pocket region [26]. The mutation is located in the D-de region of the D-E hydrophilic loop which is possibly related to the cleavage of D, in vivo. The CA1 mutant presents characteristics similar to those of DM35 (A25 IV, S264A): slow QmA reoxidation, and stabilization of the S2Q-* and SZQmB states. This phenotype could be explained by a modification of the redox couple QA/QmAthat renders the reoxidation of QwA by forward or back reactions more difficult. We concluded that in the CA1 mutant both quinone binding sites were modified. This explanation can also be used to define the phenotype of DM35 (A251V, S264A). If this is the case, not only changes in the structure of the D-de loop but also changes in the de-E part of the hydrophilic loop may induce modifications of the properties of the QA/Q-A couple in the D2 protein. DM3.5 (A25 lV, S264A) and CA1 can therefore be classified in a fourth category of mutants: Mutations which produce modifications in the QB and QA pockets inducing a stabilization of the &Q-n and S,Q-, states. Depending on their position in the D-E loop, these mutations do or do not induce herbicide resistance.

Science 115 (1996) 165-174

173

Regarding the sensitivity to high light we obviously have to group the mutants studied so far in a different way. We have already shown that the mutation A25 IV (mutation of the third type) confers to PS II an increased sensitivity to high light intensity. Under light stress, the M35 (A25 1V) mutant reaches an inactive state faster than the wild-type and from which it cannot recover its PS II activity. Double mutants presenting the A25 1V mutation plus another mutation in the Qe pocket further increase their sensitivity to high light. One of these double mutants (A25 lV, F211 S) belongs to the second category while the other (A251V, S264A) belongs to the fourth category of the previous classification. M35 (A25 1V) and AzV (A25 1V, F2 1 IS) mutants have a destabilized &Q-a while DM35 (A251V, S264A) and CA1 (also sensitive to photoinhibition) present stabilized S2QmBand SIQeA states. All these mutants have a slower fluorescence yield decay after a saturating flash, but other mutants (e.g. S264A, P255L) with similar modifications of the electron transfer kinetics do not present increased sensitivity to high light [13-151. In both double mutants, AzV (A25 lV, F211 S) and DM35 (A251V, S264A), the modifications on the donor side reactions observed in the simple mutant almost disappear. In consequence, we confirmed that there exists no obvious correlation between the apparent destabilization of S, and the sensitivity to light as we have suggested in a previous paper [ll]. The same is true for the connection between the electron transfer characteristics on the acceptor side and the high light sensitivity. There again there is no obvious correlation and our interpretation is that the rate of damage is not directly related to the stability of the Q-* state in vivo. The reason for the increased sensitivity to photoinhibition of the AzV(A251V, F21 lS), M35 (A251V), DM35 (A251V. F211S) and CA1 mutants remains an open question. References

III W.J.F. Vermaas, Photosystem II, in: L Bogorad

[21

and I.K. Vasil (Eds.), The Photosynthetic Apparatus: Molecular Biology and Operation. Academic Press, San Diego, CA, 1991, pp. 3-25. R.J. Debus. The manganese and calcium ions of photosynthetic oxygen evolution. Biochim. Biophys. Acta, 1102 (1992) 269-352.

174 [3]

[4]

[5]

[6]

[7]

[8]

[9]

[IO]

[I I]

[ 121

[ 131

[14]

[15]

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